monoclonal antibody

1. Monoclonal Antibody
Chanderhash – ASU2017010100041 | 4th Semester | School of Bioscience
BSBT 522
Pharmaceutical Biotechnology

2. Add your question here
Statement of the problem

3. Adalimumab
• Adalimumab (Humira®, AbbVie, Inc) is a recombinant human immunoglobulin (Ig)
G1 anti-tumour necrosis factor-α (TNF-α) monoclonal antibody originally approved
for the treatment of rheumatoid arthritis in the US in 2002, and subsequently
approved for the management of Crohn’s disease, ulcerative colitis, plaque psoriasis,
psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis,

4. Recombinant monoclonal antibody
• Antibody is known as immunoglobulins which are generally Y-shaped. They are
peptide molecule secreted by b cells, mostly by differentiated B cells called plasma
cells. Basically the function of antibodies is that control and stop pathogens and to
assist in an immune response. They can protect us against infection and intoxication
by mechanism of action for antibody functionality, such as antagonism, agonism, or
cell killing via ADCC, ADCP, CDC, apoptosis PCD, or lysosomal-related PCD

5. Nucleotide Synthesis
There are two pathways for synthesis of nucleotides: Salvage & de novo pathway.
Myeloma cells can only undergo de novo
pathway because they have lost the ability
to synthesise hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT)
enzyme required for Salvage Pathway.
The Aminopterin in HAT medium blocks
the enzyme required for de novo pathway.
As a result HAT medium selectively allows
growth of Hybridoma cells.

6. Production of Monoclonal Antibodies:
1. Immunization
2. Cell fusion
3. Selection of hybridomas
4. Screening the products
5. Cloning and propagation
6. Characterization and storage.

7. 1. Immunization
Antigen
Antibody producing B
cells isolated from the
mouse spleen
When the serum concentration of the antibodies is
optimal, the animal is sacrificed. The spleen is aseptically
removed and disrupted by mechanical or enzymatic
methods to release the cells. The lymphocytes of the
spleen are separated from the rest of the cells by density
gradient centrifugation.

8. 2. Cell fusion
The isolated plasma cells are fused with Myeloma cells to
create Hybridoma cells in presence of Polyethylene
Glycol (PEG).
Finally PEG is removed by washing and cells are kept inside
the fresh medium.

9. 3. Selection of hybridomas
Cells are cultured inside the HAT
medium (Hypoxanthine Aminopterin
and Thymidine). Only hybridoma
cells are grow and rest of cells are
slowly disappear within the 7-10
days. These cells are grow in regular
culture medium - produce the desired
antibody.
Unable to synthesize
DNA
1) ‘’Thymidine
kinase’’ mutation
cause a loss of
function in the
2) Aminopterin
blocks
Immortal and
restored DNA
synthesis
1) Immortally from
plasmacytoma.
2) Rescued ability to
synthesize DNA
due to restored
thymidine kinase
function.
Mortal
1) Functional DNA
synthesis, but
2) Eventually dies
because of
limited number
of replication
cycle.

10. 4. Screening the products
• Hybridomas cultured are tested for the desired antibody specificity.
• Two techniques for Screening the products:- 1) ELISA 2) RIA
• In both techniques , Antibody binds to the specific antigen and the unbound
antibody and other components of the medium can be washed off.
• Hybridoma cells producing the desired antibody can be identified by screening.
The antibody secreted by the hybrid cells is referred to as monoclonal antibody.

11. 5. Cloning and propagation
• Single hybrid cells producing the desired antibody are
isolated and cloned.
• Two techniques used for cloning hybrid cells
1) limiting dilution method
The suspension of hybridoma cells is serially diluted and
the aliquots of each dilution are put into micro culture
wells. A well contains only a single hybrid cell.
that the antibody produced is monoclonal.

12. 5. Cloning and propagation
• 2) soft agar method
• Simultaneously grow many cells in semisolid medium to form colonies.
• These colonies will be monoclonal in nature.
• Both the above techniques are combined and used for maximal production of MAbs.

13. 6. Characterization and storage.
• The monoclonal antibody has to be subjected to biochemical and biophysical
characterization for the desired specificity.
• It is also important to elucidate the MAb for the immunoglobulin class or sub-class,
the epitope for which it is specific.
• The stability of the cell lines and the MAbs are important.
• The cells (and MAbs) must be characterized for their ability to withstand freezing,
and thawing.
• The desired cell lines are frozen in liquid nitrogen at several stages of cloning and
culture.number of binding sites it possesses.

14. Production of Mabs in E. coli
mRNA from B-lymphocytes
cDNA
PCR amplifies Heavy and Light chain
Cutting by restriction endonucleases enzyme
H-chain DNA sequence L-chain DNA sequence
Cloned in vector cloned in vector
H and L chains combined
And cloned in vector
Screen for antigen binding
Incorporate H and L sequence
in a plasmid
E.Coli transformed with H.L DNA construct
Production of Fv fragment in E.coli
The objective is to develop
bioreactors for the large
scale production of
monoclonal antibodies.
Production of
bispecific
monoclonal
antibody

15. Major limitations:
• i. For ethical reasons, humans cannot be immunized against antigens.
• ii. The fused human lymphocyte-mouse myeloma cells are very unstable.
• iii. There are no suitable myeloma cells in humans that can replace mouse myeloma
cells.
• The presence of retroviruses as a part of the mammalian chromosomes is a common
occurrence. Mice used in MAb production carry several viruses (adenovirus, hepatic
virus, retrovirus, reovirus, cytomegalovirus, thymic virus). The presence of some of
these viruses has been detected in the hybridomas.

16. Monoclonal for tumour therapy
• Cell Depletion
Rituxan,Campath (naked)
Myelotarg(drug)
• Blocking receptors
Herceptin
• Attacking vasculature
Avastin,Erbitux
• Vaccination against idiotype
Panorex

17. Advantages of Monoclonal Antibodies:
• Monoclonal antibodies truly represent a homogeneous state of a single molecular
species.
• They are specific to a particular antigen with a particular epitopes.
• For example. Rituximab (rituxan, anti-CD20) is a good antibody for the treatment of
Lymphoma.

18. Conclusion…
Monoclonal antibodies have a wide range of applications from diagnostic (Western Blot,
Immunohistochemistry) to therapeutic (oncology, autoimmune disease).
Immuno-Oncology has a great potential in providing more effective treatment in cancer types
where none exists.
It also paves the way for personalized medicine.
In Feb 2014, Empliciti (Elotuzumab) was granted “Breakthrough Therapy” in the treatment of
Multiple Myeloma by the FDA.
While there are some limitations, the potential of mAb is enormous.

19. Reference ..
• https://www.britannica.com/science/monoclonal-antibody
• National Research Council (US) Committee on Methods of Producing
Monoclonal. Monoclonal Antibody Production.
https://www.ncbi.nlm.nih.gov/books/NBK100191/
• https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/
• file:///C:/Users/Dell/Desktop/Article%20017.pdf
• http://www.molecular-plant-biotechnology.info/hybri doma-and-monoclonal-
antibodies-mabs/ raising-theantibodies.htm